Synthesis, antitumor activity and CDK1 inhibiton of new thiazole nortopsentin analogues

Article abstract of DOI:10.1016/j.ejmech.2017.06.052

A new series of thiazole nortopsentin analogues was conveniently synthesized with fair overall yields. The antiproliferative activity of the new derivatives was tested against different human tumor cell lines of the NCI full panel. Four of them showed good antitumor activity with GI₅₀ values from micro to nanomolar level. The mechanism of the antiproliferative effect of these derivatives, was pro-apoptotic, being associated with externalization of plasma membrane phosphatidylserine and DNA fragmentation. The most active and selective of the new thiazoles confined viable cells in G2/M phase and markedly inhibited in vitro CDK1 activity.

Full text of DOI:10.1016/j.ejmech.2017.06.052

Accepted Manuscript
Synthesis, antitumor activity and CDK1 inhibiton of new thiazole nortopsentin
analogues
Barbara Parrino, Alessandro Attanzio, Virginia Spanò, Stella Cascioferro, Alessandra
Montalbano, Paola Barraja, Luisa Tesoriere, Patrizia Diana, Girolamo Cirrincione,
Anna Carbone
PII:
S0223-5234(17)30506-8
10.1016/j.ejmech.2017.06.052
EJMECH 9547
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 April 2017
Revised Date: 24 May 2017
Accepted Date: 25 June 2017
Please cite this article as: B. Parrino, A. Attanzio, V. Spanò, S. Cascioferro, A. Montalbano, P. Barraja,
L. Tesoriere, P. Diana, G. Cirrincione, A. Carbone, Synthesis, antitumor activity and CDK1 inhibiton of
new thiazole nortopsentin analogues, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.06.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, antitumor activity and CDK1 inhibiton of new thiazole nortopsentin analogues
Barbara Parrino, Alessandro Attanzio, Virginia Spanò, Stella Cascioferro, Alessandra Montalbano,
Paola Barraja, Luisa Tesoriere, Patrizia Diana, Girolamo Cirrincione, Anna Carbone*
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo (Italy).
Corresponding author. Tel.: +39-091-6161606; +39-091-23896826; E-mail address:
anna.carbone@unipa.it

ACCEPTED MANUSCRIPT
Abstract
A new series of thiazole nortopsentin analogues was conveniently synthesized with fair overall
yields. The antiproliferative activity of the new derivatives was tested against different human
tumor cell lines of the NCI full panel. Four of them showed good antitumor activity with GI50
values from micro to nanomolar level. The mechanism of the antiproliferative effect of these
derivatives, was pro-apoptotic, being associated with externalization of plasma membrane
phosphatidylserine and DNA fragmentation. The most active and selective of the new thiazoles confined
viable cells in G2/M phase and markedly inhibited in vitro CDK1 activity.
Keywords: Marine alkaloids, nortopsentin analogues, antiproliferative activity, apoptosis, CDK1
inhibitors, thiazolyl-1H-pyrrolo[2,3-b]pyridines.

ACCEPTED MANUSCRIPT
1. Introduction
In recent years, many efforts have been done to develop new small molecules active as antitumor
agents [1-7] as well as to discovery new targets for cancer treatment. Human kinases remain
interesting targets in oncology [8] and, among 518 human kinases, the family of cyclin-dependent
kinases (CDKs) has attracted considerable interest due to their involvement in several pathological
processes such as cancer [9]. Marine environment is an important source of secondary metabolites,
among them numerous alkaloids acting as CDK inhibitors.
Variolins, isolated from the rare Antarctic sponge Kirkpatrickia variolosa, are marine alkaloids with
an unique pyrido[3’,2’:4,5]pyrrolo[1,2-c]pyrimidine core (Chart 1). Four variolins were isolated:
variolin A, variolin B, N(3’)-methyltetrahydrovariolin B and variolin D [10,11]. Variolin B and its
synthetic analogue, deoxyvariolin B, showed in vitro activity against P388 cell line with IC50 values
of 210 nM and 157 nM respectively [12]. The latter compounds caused cell cycle perturbations,
induced apoptosis and inhibited different cyclin-dependent kinases, in particular CDK1 and CDK2,
in the micromolar range [13].
Meridianins A-G are a family of brominated and/or hydroxylated 3-(2-aminopyrimidine)indoles,
which are purified from Aplidium meridianum, an ascidian collected in the South Atlantic [14]
(Chart 1). Also these alkaloids prevented cell proliferation, induced apoptosis and resulted potent
inhibitors of several protein kinases. In particular, meridianin E showed potent and selective
inhibiton of CDK1 and CDK5 in the low micromolar range [15,16].
A new class of 3-(pyrimidin-4-yl)-7-azaindoles, meriolins (Chart 1), was synthesized as a chemical
hybrid between the natural compounds variolins and meridianins. These new derivatives showed
better antitumor and proapoptotic properties in human tumor cell cultures than their parent
compounds and a relative selectivity against CDKs, especially CDK1, CDK2 and CDK9 [17,18].

ACCEPTED MANUSCRIPT
Me
H2N
H2N
N
N
N
NH2
Me
N
N
OH
R
N
OH
N
N
R
COOMe
O
N
N
N
N
N
N
N
N
N
N
H2N
H2N
H2N
H2N
Variolin B
Variolin A
R = OH
N(3')-methyltetrahydrovariolin B
Variolin D
Deoxyvariolin B R = H
N
N
NH2
NH2
R
N
N
R
N
R1
R2
N
N
H
R1
R2
R3
Meriolins
Meridianin A R = OH; R1 = R2 = R3 = H
Meridianin B R = OH; R1 = R3 = H; R2 = Br
Meridianin C R = R2 = R3 = H; R1 = Br
Meridianin D R = R1 = R3 = H; R2 = Br
Meridianin E R = OH; R1 = R2 = H; R3 = Br
Meridianin F R = R3 = H; R1 = R2 = Br
Meridianin G R = R1 = R2 = R3 = H
R = H, Cl, OMe, OEt, OPr, OiPr, O(CH2)2OMe
R1 = H, Br
R2 = H, Me, SO2Ph
Chart 1. Marine alkaloids active as CDK1 inhibitors.
Nortopsentins A-C (Chart 2), isolated from the Caribbean deep sea Spongosorites ruetzleri, are bis-
indolyl alkaloids, having an imidazole as a spacer between the two indole units, with an unknown
mechanism of action. They displayed in vitro cytotoxicity against P388 cells (IC50 4.5-20.7 µM)
and their N-methylated derivatives showed a significant improvement in P388 activity compared to
that of the parent compounds (IC50 0.8-2.1 µM) [19,20]. Due to its interesting antitumor activity,
nortopsentin has been considered an important lead compound for the design of new biologically
active derivatives. Analogues in which the imidazole ring of the alkaloid was replaced by other five
membered heterocycles such as bis-indolyl-thiophenes [21], -thiazoles [22], -pyrazoles [23], -furans
and isoxazoles [24], -pyrroles [25], and -1,2,4-thiadiazoles [26] were reported. Most of them
showed antiproliferative activity against a wide range of human tumor cell lines with GI50 in the
micromolar to sub-micromolar range. In literature are reported many analogues, in which beside the
imidazole core, one or both indole units were replaced by phenyl, azaindole or naphthyl moieties
[27-33]. In particular, 3-[(2-indolyl)-5-phenyl]-pyridines 1 [27] and phenyl-thiazolyl-7-azaindoles 2
(Chart 2) [28] showed cytotoxic activity in the micro-nanomolar range and inhibited CDK1 with
IC50 values of 0.31-0.90 and 0.41-0.85 µM, respectively. Indolyl-thiazolyl-4-azaindole 3 and
indolyl-thiazolyl-7-azaindole 4 derivatives (Chart 2) were active against a wide range of human

ACCEPTED MANUSCRIPT
tumor cell lines in the micro-submicromolar range, including STO and Meso II cell lines derived
from human diffuse malignant peritoneal mesothelioma, a rapidly lethal human malignancy
generally refractory to currently available therapies. These azaindole analogues were also able to
inhibit CDK1 with IC50 values of 0.64-0.89 µM, comparable to those reported for roscovitine and
purvalanol A, two well-known CDK1 inhibitors [29,30]. Moreover, in the mouse model,
intraperitoneal administration of some derivatives of type 4 resulted in a significant tumor volume
inhibition of DMPM xenografts (58-75%) at well-tolerated doses, and two complete responses were
observed in each treatment group [30].
NH
N
R
R2
R3
R
N
N
N
R2
R1
R1
N
Nortopsentin A R = R3 = Br; R1 = R2 = H
Nortopsentin B R = R1 = R2 = H; R3 = Br
Nortopsentin C R = Br; R1 = R2 = R3 = H
1
R
S
S
R
X
R3
R1
R2
N
N
Y
N
N
N
2
N
R2
R1
R3
3 X = N; Y = CH
4 X = CH; Y = N
S
R
N
N
N
N
5-7
R2
R1
Chart 2. Nortopsentins and their analogues able to inhibit CDK1.
Continuing our studies on nitrogen heterocyclic systems endowed with antitumor activity [34-41],
herein we report a new series of thiazoles 5-7 (Chart 2), analogues of derivatives 4, in which the
nitrogen atom of the indole and/or 7-azaindole moiety is decorated with a 2-methoxyethyl chain
with the aim of increasing the water solubility. We also describe the cytotoxic activity of the new
derivatives against different human tumor cell lines as well as their capacity to inhibit CDK1.

ACCEPTED MANUSCRIPT
2. Chemistry
Our plan for the synthesis of new substituted thiazoles 5a-q and 6a,b was based on the Hantzsch
reaction between thioamides 16d, 17a-d, 18a-d and 19a-c and α-bromoacetyl derivatives 21a-c
(Scheme 1). Indole-3-carbothioamides 16d, 18a-d and 19a-c were obtained from the corresponding
indoles 8d, 10a-d, and 11a-c through the formation of indole-3-carboxamides 12d, 14a-d and 15a-c
as previously reported [30].
The new thioamides 17a-d were conveniently synthesized from the corresponding amides 13a-d in
turn prepared (65-98% yields) by reaction of 1-(2-methoxyethyl)-1H-indoles 9a-d with
chlorosulfonyl isocyanate (CSI) in acetonitrile at 0 °C followed by alkaline hydrolysis of the
chlorosulfonyl group. The subsequent reaction of new amides 13a-d with Lawesson’s reagent under
reflux in tetrahydrofuran (THF) gave the desired thioamides 17a-d in very good yields (70-99%)
(Scheme 1).
3-Bromoacetyl-7-azaindoles 21a,c were obtained from the corresponding 7-azaindoles 20a,c as
previously reported [28]. The new 2-bromo-1-[1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridin-3-
yl]ethanone 21b was efficiently synthesized (70% yield) by reaction of 1-(2-methoxyethyl)-1H-
pyrrolo[2,3-b]pyridine 20b with bromoacetyl bromide in presence of aluminum chloride in
dichloromethane (Scheme 1).
1-(2-Methoxyethyl)-1H-indoles 9a-d and 1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridine 20b were
obtained in excellent yields (81-99%) from the corresponding indoles 8a-d or azaindole 20a by
alkylation with 2-bromoethyl methyl ether using N,N-dimethylformamide (DMF) as a solvent and
sodium hydride as base. [42,43]
The final reaction between the appropriate thioamides 16d, 17a-d, 18a-d and 19a,c and the suitable
α-bromoacetyl compounds 21a-c in ethanol under reflux gave the desired new thiazoles 5a-q and
6a,b (Table 1) in good to excellent yields (56-98%). The subsequent deprotection of N-tert-
butylcarboxylate derivatives 6a,b by using trifluoroacetic acid (TFA) in dichloromethane (DCM)
under reflux afforded, after neutralization, the corresponding N-deprotected thiazoles 7a,b in good
yields (60,72%) (Table 1). The reaction of thioamide 19b with the 3-bromoacetyl derivative 21b
gave a very instable compound unsuitable to undergo biological assay. The crude of this latter
compound was used for the next N-Boc deprotection but also in this case an impure deprotected
derivative was obtained.

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O
S
NH2
R
NH2
R
v
N
N
R1
12d R1 = H
13a-d R1 = CH2CH2OMe
14a-d R1 = Me
R1
16d R1 = H
17a-d R1 = CH2CH2OMe
18a-d R1 = Me
19a-c R1 = Boc
15a-c R1 = Boc
iv
R
S
viii
N
R
N
N
N
R1
R2
N
5a-q
6a,b
7a,b
R1
a R = H, b R = OMe, c R = Br, d R = F
ix
8a-d R1 = H
i
ii
iii
9a-d R1 = CH2CH2OMe
10a-d R1 = Me
11a-c R1 = Boc
O
Br
vii
N
N
N
N
R2
R2
21a R2 = H
21b R2 = CH2CH2OMe
21c R2 = Me
20a R2 = H
20b R2 = CH2CH2OMe
20c R2 = Me
i
vi
Scheme 1. Synthesis of new thiazole derivatives 5a-q, 6a,b and 7a,b.
Reagents: (i) (a), NaH, DMF, 0 °C-rt, 30 min; (b) BrCH2CH2OMe, 60 °C, 24 h, 81-99%; (ii) and
(iii) for compounds 10a-d and 11a-c: see reference [30]; (iv) for compounds 12d, 14a-d and 15a-c:
see reference [30]; for compounds 13a-d: (a) CSI, MeCN, 0 °C, 30 min; (b) 10% KOH, aq acetone,
65-98%; (v) for compounds 16d, 18a-d and 19a-c: see reference [30]; for compounds 17a-d:
Lawesson’s reagent, THF, reflux, 30 min, 70-99%; (vi) for compound 20c: see reference [28]; (vii)
for compounds 21a,c: see reference [28]; for compound 21b: AlCl3, DCM, BrCH2COBr, reflux, 40
min, 70%; (viii) EtOH, reflux, 30 min, 56-98%; (ix) (a) TFA, DCM, reflux, 24 h; (b) aq NaHCO3,
60,72%.

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Table 1. New thiazole derivatives 5a-q, 6a,b and 7a,b.
R
S
N
N
N
N
R1
R2
Compd
5a
R
H
H
H
H
R1
R2
H
Me
Compd
5l
5m
5n
R
Br
F
F
F
F
F
H
Br
H
Br
R1
Me
R2
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe CH2CH2OMe
H
Me
5b
5c
5d
5e
5f
5g
5h
5i
5j
Me
CH2CH2OMe
5o
5p
5q
6a
6b
7a
7b
OMe CH2CH2OMe
OMe CH2CH2OMe
OMe CH2CH2OMe CH2CH2OMe
OMe
Br
Br
H
Me
Me
H
Boc
Boc
H
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
Me
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
H
Me
H
Br
CH2CH2OMe CH2CH2OMe
5k
3. Results and Discussion
3.1 Cytotoxic activity
All the synthesized thiazoles 5a-q, 6a,b and 7a,b were submitted to the National Cancer Institute
(NCI; Bethesda, MD), and derivatives 5a-h, l, o-q, 6a,b and 7a,b were prescreened according to the
NCI protocol at a 10−5 M dose (data not shown) on the full panel of approximately 60 human cancer
cell lines derived from 9 human cancer cell types that have been grouped into disease subpanels
including leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal,
prostate, and breast tumor cell lines. Compounds 5e,q and 7a,b satisfied the criteria set by the NCI
for activity in this assay and were selected for further screenings at five concentrations at 10-fold
dilution (10−4−10−8 M) on the full panel. The growth inhibition activity of compounds was defined
in terms of the GI50 value (which represents the molar concentration of the compound that inhibits
50% net cell growth). The average values of the mean graph midpoint (MG_MID) was calculated.
Data reported in Table 2 revealed that thiazoles 5e,q and 7a,b showed antiproliferative activity in
the micromolar to nanomolar range (GI50 0.03-98.0 µM). Compounds 5e and 7b were active against
the total number of cell lines investigated, whereas compounds 5q and 7a were cytotoxic against a
very high percentage of the tested cell lines (96% and 93% respectively). The most active
compound was thiazole 5e (MG_MD 3.6 µM) followed by thiazoles 7b, 7a and 5q (MG_MD 4.5,
5.4 and 6.3 µM respectively).

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Table 2. Overview of the in vitro antitumor screening[a] results for compounds 5e,q and 7a,b.
[b]
GI50
[c]
Compd
5e
Ncl
N[d]
57
51
51
54
Range
MG_MID[e]
57
0.03-12.6
0.65-98.0
0.42-28.2
1.79-13.9
3.6
6.3
5.4
4.5
53
55
54
5q
7a
7b
[a] Data obtained from the NCI in vitro disease-oriented human tumor cell line screen.
[b] Concentration (µM) that inhibits 50% net cell growth. [c] Number of cell lines
investigated. [d] Number of cell lines giving positive GI50 values. [e] MG_MID: mean
graph midpoint; this is the arithmetic mean value for all tested cancer cell lines.
Derivative 5e was particularly efficacious against the breast cancer sub-panel (Table 3) having GI50
in the range 0.03-2.38 µM. The most sensitive breast cancer cell lines were MDA-MB-468 (GI50
0.03 µM), T-47D (GI50 0.04 µM) and MCF-7 (GI50 0.05 µM). Moreover, thiazole 5e exhibited good
selectivity with respect to the non-small cell lung cancer (GI50 0.07-5.18 µM) and ovarian cancer
(GI50 0.03-4.66 µM) sub-panels and to the KM12 (GI50 0.16 µM) of the colon cancer sub-panel.
Thiazoles 5q and 7a (Table 3) showed good selectivity against KM12 cell line (GI50 1.77 and 0.76
µM respectively) of the colon cancer sub-panel, MDA-MB-435 cell line (GI50 0.90 and 0.86 µM,
respectively) of the melanoma sub-panel and T-47D cell line (GI50 0.65 and 0.42 µM, respectively)
of the breast cancer sub-panel.
In spite of the low number of derivatives that underwent the test on the NCI full panel of human cell
lines, it is possible to outline some SAR. The most active compound is 5e (MG_MD 3.6 µM) which
bears an unsubstituted azaindole portion, a methoxy group at position 5 and a methyloxyethyl
substituent at the imino nitrogen of the indole moiety. The introduction of a methyl or a
methyloxyethyl group at the imino nitrogen of the azaindole moiety as well as the replacement of
the methoxy group with a hydrogen led to inactive compounds (5f, 5g and 5a did not satisfied
criteria to be tested on the full panel). Moving the methyloxyethyl substituent from the indole to the
azaindole moiety maintained the activity, although slightly lower. However, also in this series the
introduction of a methyl group on the imino nitrogen of the indole moiety dropped the activity (5d,
5l and 5p did not satisfied criteria to be tested on the full panel).

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Table 3. In vitro inhibition (µM) of cancer cell line growth by compounds 5e,q and 7a,b.
[a]
[a]
Cell line
GI50
Cell line
GI50
5e
5q
7a
7b
5e
5q
7a
7b
Leukemia
CCRF-CM
K-562
Melanoma
LOX IMVI 3.69
MALME-
3M
6.70
4.79
-
>100
2.80
-
3.44
4.54
3.96
3.58
2.67
3.66
2.12
3.60
12.6
MOLT-4
RPMI-8226
6.02
7.09
5.56
-
-
-
3.87
5.14
M14
5.82
3.14
0.90
3.22
0.86
3.27
4.12
MDA-MB- 6.41
435
SR
1.28
3.24
3.02
3.25
SK-MEL-2 3.56
3.89
7.72
7.38
8.19
6.82
4.79
SK-MEL-
28
7.39
Non-Small
Cell Lung
Cancer
A549/ATCC 4.41
4.51
1.98
2.02
6.29
7.10
2.00
2.82
14.9
6.89
2.05
7.98
4.39
SK-MEL-5 5.53
UACC-257 4.26
2.69
-
6.78
3.22
>100
4.58
3.24
8.69
4.20
EKVX
HOP-62
HOP-92
0.07
2.12
0.99
UACC-62
Ovarian
Cancer
5.98
NCI-H23
NCI-H322M 1.95 >100
NCI-H460
NCI-H522
Colon
1.63
3.88
4.58
>100
3.40
2.07
3.21
11.8
3.07
2.17
IGROV1
0.55
3.49
3.34
2.59
10.1
4.10
2.53
2.94
1.76
28.2
8.00
4.06
3.90
2.74
4.83
5.35
OVCAR-3 4.66
OVCAR-4 0.03
OVCAR-5 0.61
OVCAR-8 4.11
1.32
5.18
3.38
2.19
Cancer
COLO-205
5.03
2.69
2.66
3.22
NCI/ADR- 3.07
RES
2.17
-
1.76
14.8
2.86
13.9
HCC-2998
HCT-116
4.81
4.55
4.54
3.77
4.08
3.63
3.80
3.84
SK-OV-3
Renal
3.49
Cancer
786-0
A498
HCT-15
HT29
KM12
2.15
4.31
0.16
4.50
2.35
2.90
1.77
3.63
2.34
3.19
0.76
3.52
3.14
3.36
2.74
3.30
3.44
1.87
3.08
4.36
1.18
5.51
2.31
5.12
3.26
5.27
9.54
5.91
>100
4.41
6.25
3.03
5.60
8.36
5.76
>100
4.15
4.03
3.02
3.04
5.21
3.30
7.72
3.10
ACHN
CAKI-1
RXF 393
SN12C
UO-31
Breast
Cancer
MCF-7
SW-620
CNS Cancer
SF-268
4.42
2.70
3.99
7.67
3.20
8.86
6.98
3.35
5.91
-
SF-295
SF-539
2.83
7.34
SNB-19
SNB-75
9.71
1.10
98.0
2.97
16.2
1.43
12.2
4.72
0.05
2.09
2.59
1.74
5.05
1.79
2.65
MDA-MB- 2.02
231/ATCC
U251
Prostate
Cancer
4.43
5.14
6.12
6.18
HS 578T
BT-549
2.07
2.38
8.82
4.26
9.14
4.45
4.44
4.80
T-47D
MDA-MB- 0.03
468
0.04
0.65
2.44
0.42
0.97
1.97
2.34
PC-3
4.22
4.28
5.06
18.8
6.81
15.4
3.68
-
DU-145
[a] Concentration (µM) that inhibits 50% net cell growth.

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3.2 Cell apoptosis
To gain insight into the mechanism of growth inhibitory effects (necrosis or apoptosis) of the
synthesized thiazoles 5e,q and 7a,b, flow cytometry analysis of annexin V-FITC and propidium
iodide (PI) stained MCF-7 cells was carried out to evaluate externalization of plasma membrane
phosphatidylserine, a reliable marker of cell apoptosis. The chosen concentrations were selected
taking into account the GI50 values measured at 72 h for this cell line, i.e. 5x10-8 M for 5e and 2x10-
6 M for 5q,7a and 7b (Table 3). As shown in Figure 1, none of the nortopsentin analogues exerted
necrotic effects, while induced a clear shift of viable cells towards early apoptosis. In addition, the
percentage of apoptotic cells after treatment with compound 5e (54.2%) was higher than that
displayed by the other analogues.
Figure 1. Flow cytometric analysis for the quantification, by annexin V/PI double staining, of
nortopsentin analogues induced apoptosis in MCF-7 cells. Cell monolayers were incubated for
72 h in the absence (control) or in the presence of the synthesized compounds at their relevant GI50
values and submitted to double staining with annexin V/PI as reported in Paragraph 4.2.2. BU3,
viable cells (annexin V-/P-); BU 4, cells in early apoptosis (annexin V+/PI-); BU 2, cells in tardive
apoptosis (annexin V+/PI+); BU E1, necrotic cells (annexin V-/PI+). Representative images of
three experiments with comparable results.

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3.3 Cellular cycle analysis
One main feature of cancer cells is uncontrollable replication, and then cell cycle arrest become an
important target in oncotherapy. Cell cycle distribution in PI stained MCF-7 cells was evaluated by
flow cytometry. The analysis was performed after 48 h of incubation with tested compounds in
order to detect the shifts in cell cycle distribution before a significant amount of cells underwent
apoptosis. Appearance of a sub-G1-cell population, which is representative of cells with fragmented
DNA, was evident after treatment with each of the compounds and it is consistent with their
apoptotic activity (Figure 2). Moreover, derivatives 5e,q and 7a caused a net accumulation of cells
in G2/M phase with a concomitant percentage reduction of cells in both the S and G0/G1 phase.
Among all derivatives, 5e appeared the most effective, confining more than 50% of the viable cells
in G2/M phase. Compound 7b, on the contrary, produced a moderate increase (9%) of viable cells
in G0/G1 compared to the control (63.5%). These data suggested that 5e,q and 7a produced mitotic
failure on MCF-7 cells, while thiazole 7b weakly affected the G1/S transition of cell cycle.
120
G2/M
S
G0/G1
80
40
0
subG0/G1
control
5e
5q
7a
7b
Figure 2. Cell cycle analysis of MCF-7 cells treated with compounds 5e,q and 7a,b. Cell
monolayers were incubated in the absence (control) or in the presence of the compounds at their
relevant GI50 values. After 48 h incubation, propidium iodide-stained cells were submitted to flow
cytometric analysis as reported in Paragraph 4.2.3. The percentage of cells in the different phases
of the cycle was calculated by Expo32 software. Values are the mean±SD of three separate
experiments in triplicate.
3.4 Selective cytotoxicity
In order to determine the selective cytotoxicity of derivatives 5e,q and 7a,b, additional experiments
were performed on intestinal normal-like differentiated Caco-2 cells. While thiazole 5e did not

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affect the cell viability at any assayed concentration, a reduction of the cell survival was detected
after treatment with compounds 5q and 7a,b in the 25-100 µM range (Figure 3).
100
75
5e
50
5q
7a
25
7b
0
0
25
50
75
100
Concentration (µM)
Figure 3. Effect of compunds 5e,q and 7a,b on the viability of human intestinal normal-like
differentiated Caco-2 cells. Cells were treated with the compounds and cell viability was measured
after 72 h by MTT assay in comparison to cells treated with vehicle alone (control). Values are the
mean±SD of three separate experiments in triplicate.
Table 4 reports the calculated LC50 values and the selectivity index (SI). The high values of SI
indicated that the synthesized compounds are selectively cytotoxic to cancer cells, suggesting their
potential use as antiproliferative agents.
Table 4. Cytotoxic activity and the selectivity index (SI) of the synthesized compounds 5e,q and
7a,b against MCF-7 cancer cell line.
Normal-like intestinal cells
MCF-7
GI50 (µM)a
SI
LC50 (µM)
5e
5q
7a
7b
<100
0.05
2.09
1.74
1.79
nd
46
41
46
96.1±3.1
71.4±2.1
83.3±2.5
LC50 concentration which is lethal to 50% of the normal cells compared to untreated controls; GI50 concentration
required to inhibit the tumor cell growth by 50% compared to untreated controls; nd, not determined.
aData obtained from the NCI.

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3.5 CDK1 activity inhibition
Since azaindole nortopsentin analogues have been reported to effectively inhibit CDK1 activity and
consequently induce cell cycle arrest at the G2/M phase [28-30], we assessed whether the thiazole
derivative 5e, the new most active and highly selective derivative against human cancer cells, could
inhibit the in vitro catalytic activity of CDK1. For comparison, we decided to use an indolyl-
thiazolyl-7-azaindole derivative of type 4 (Chart 2), i.e. (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-
1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b] pyridine (positive control), previously synthesized by
our research group and known to act as a potent inhibitor of CDK1 activity [30]. We also evaluated
compound 7b as an expected negative control. Nortopsentin derivative 5e in the range 0.1-10 µM
markedly inhibited CDK1 activity, with IC50 value of 1.14±0.09 µM, not significantly different
(p>0.05, Student’s t-test) from that calculated, in the same condition, for the positive control
(0.97±0.01) (Figure 4). On the other hand, derivative 7b did not affect the enzyme activity (Figure
4).
15
5e
7b
10
5
positive control
0
0.1
1
10
Inhibitor (µM)
Figure 4. Inhibition of CDK1/cyclin B activity. The activity of recombinant human CDK1/cyclin
B was assayed using Omnia® Kinase Assay Kit as reported in Paragraph 4.2.4, in the presence of
increasing concentration of compounds either 5e, 7b and positive control. Initial velocity of the
reaction (RCFU/s) was determined and plotted against compound concentration to calculate IC50
value. Values are the mean±SD of two determinations.
4. Conclusions
A new series of thiazole nortopsentin analogues of type 5-7 in which the nitrogen atom of the indole
and/or 7-azaindole moiety is decorated with a 2-methoxyethyl chain was efficiently synthesized.
Four of the new nortopsentin derivatives, 5e,q and 7a,b showed good antiproliferative activity
against almost the totality of the about 60 human tumor cell lines of NCI full panel. The mechanism

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of their antiproliferative effect, investigated on human breast cancer MCF-7 cells, was pro-
apoptotic, being associated with externalization of plasma membrane phosphatidylserine and DNA
fragmentation, accompanied to perturbation of the cell cycle progression. With the exception of
compound 7b, the newly synthesized nortopsentin derivatives confined viable cells in G2/M phase.
Derivative 5e showed the most interesting in vitro anticancer activity, expressing lower GI50 values,
ranging from low micromolar to nanomolar level (12.6-0.03 µM), high selectivity towards tumor
cells and high efficacy in causing mitotic failure. Importantly, compound 5e markedly in vitro
inhibited CDK1 activity with IC50 value comparable to that reported for other indolyl-thiazolyl-7-
azaindole derivative or well-known CDK1 inhibitors, roscovitine and purvalanol A [30]. Overall,
our data indicated that a possible growth inhibitory mechanism induced on MCF-7 cells by 5e, was
CDK1/cyclin B inhibition-induced G2/M phase arrest. Decoration of the nitrogen atom of the indole
and/or 7-azaindole moiety with 2-methoxyethyl chain led to interesting biological results. For this
reason, further developments of this class of compounds through decoration with other suitable
chains should be encourage.
5. Experimental
5.1 Chemistry
All melting point were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR spectra
were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1H and 13C NMR
spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 solution, using a Bruker
Avance II series 200 MHz spectrometer. Column chromatography was performed with Merk silica
gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge
system). Elemental analyses (C, H, N) were within ± 0.4% of theoretical values and were performed
with a VARIO EL III elemental analyzer. Purity of all the tested compounds was greater than 95%,
determined by HPLC (Agilent 1100 Series). Mass spectra were obtained using a MarinerTM mass
spectrometer, Applied Biosystems (Foster City, CA). A Harvard model 11 syringe pump (Holliston,
MA) was used to infuse the sample solutions. The ESI source was operated in positive ion mode
with an electrospray voltage of 4.5 kV.
1H and 13C NMR spectra of compounds 9b, 20b, 13b, 17b, 21b, 5e, 5q, 6a, 6b, 7a and 7b are
reported as examples in the supporting informations.
General procedures, analytical and spectroscopic data for compounds (10a-d), (11a-c), (12d), (14a-
d), (15a-c), (16d), (18a-d), (19a-c), (20c) and (21a,c) were previously reported [28,30].

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5.1.1 General Procedure for the Synthesis of 1-(2-methoxyethyl)-1H-indoles (9a-d) and 1-(2-
methoxyethyl)-1H-pyrrolo[2,3-b]pyridine (20b)
To a cold solution of the appropriate indole 8a-d or azaindole 20a (2.6 mmol) in anhydrous DMF
(7.8 mL) NaH (60% suspension in mineral oil, 3.9 mmol, 0.16 g) was added. After 30 min stirring
at room temperature, 2-bromoethyl methyl ether (3.1 mmol, 0.3 mL) was added. The reaction
mixture was heated at 60 °C for 24 h. After cooling, the mixture was poured into ice-water and
extracted with ethyl acetate (3 x 20 mL). The organic phases were dried (Na2SO4) and evaporated
under reduced pressure. The residue was used in the next step without any further purification (for
compounds 9b-d) or purified by column chromatography using petroleum ether/dichloromethane
(65/35) (for compound 9a) or petroleum ether/ethyl acetate (30/70) (for compound 20b) as eluent.
5.1.1.1 1-(2-Methoxyethyl)-1H-indole (9a)
1
Yellow oil; yield: 83%; H NMR (200 MHz, DMSO-d6) δ: 3.21 (s, 3H, CH3), 3.64 (t, 2H, J = 5.3
Hz, CH2), 4.33 (t, 2H, J = 5.3 Hz, CH2), 6.43 (d, 1H, J = 3.1 Hz, H-3), 6.99-7.17 (m, 2H, H-5 and
13
H-6), 7.35 (d, 1H, J = 3.1 Hz, H-2), 7.47-7.57 (m, 2H, H-4 and H-7); C (50 MHz, DMSO-d6) δ:
45.2 (t), 58.0 (q), 71.0 (t), 100.4 (d), 109.8 (d), 118.8 (d), 120.3 (d), 120.9 (d), 128.0 (s), 129.0 (d),
135.8 (s). Anal. Calcd for C11H13NO: C, 75.40; H, 7.48; N, 7.99. Found: C, 75.70; H, 7.33; N, 7.82.
MS m/z 176 (MH+). HRMS: [MH]+, found 176.1089 C11H13NO requires 176.1070.
5.1.1.2 5-Methoxy-1-(2-methoxyethyl)-1H-indole (9b)
1
Yellow oil; yield: 99%; H NMR (200 MHz, DMSO-d6) δ: 3.21 (s, 3H, CH3), 3.62 (t, 2H, J = 5.0
Hz, CH2), 3.75 (s, 3H, CH3), 4.28 (t, 2H, J = 5.0 Hz, CH2), 6.34 (d, 1H, J = 2.2 Hz, H-3), 6.78 (dd,
1H, J = 8.8, 2.0 Hz, H-6), 7.05 (d, 1H, J = 2.0 Hz, H-4), 7.30 (d, 1H, J = 2.2 Hz, H-2), 7.38 (d, 1H,
13
J = 8.8 Hz, H-7); C (50 MHz, DMSO-d6) δ: 45.4 (t), 55.2 (q), 58.0 (q), 71.1 (t), 100.1 (d), 101.9
(d), 110.5 (d), 111.0 (d), 128.4 (s), 129.4 (d), 131.1 (s), 153.3 (s). Anal. Calcd for C12H15NO2: C,
70.22; H, 7.37; N, 6.82. Found: C, 69.96; H, 7.52; N, 7.03. MS m/z 206 (MH+). HRMS: [MH]+,
found 206.1152 C12H15NO2 requires 206.1176.
5.1.1.3 5-Bromo-1-(2-methoxyethyl)-1H-indole (9c)
1
Yellow oil; yield: 99%; H NMR (200 MHz, DMSO-d6) δ: 3.20 (s, 3H, CH3), 3.63 (t, 2H, J = 5.0
Hz, CH2), 4.33 (t, 2H, J = 5.0 Hz, CH2), 6.43 (d, 1H, J = 2.3 Hz, H-3), 7.23 (d, 1H, J = 8.7 Hz, H-
13
6), 7.71 (d, 1H, J = 2.3 Hz, H-2), 7.48 (d, 1H, J = 8.7 Hz, H-7), 7.73 (s, 1H, H-4); C (50 MHz,
DMSO-d6) δ: 46.4 (t), 59.0 (q), 71.9 (t), 101.1 (d), 112.4 (s), 112.9 (d), 123.3 (d), 124.2 (d), 130.8
(s), 131.5 (d), 135.5 (s). Anal. Calcd for C11H12BrNO: C, 51.99; H, 4.76; N, 5.51. Found: C, 51.75;

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H, 4.62; N, 5.70. MS m/z 254 (MH+). HRMS: [MH]+, found 254.0148 C11H12BrNO requires
254.0175.
5.1.1.4 5-Fluoro-1-(2-methoxyethyl)-1H-indole (9d)
1
Yellow oil; yield: 98%; H NMR (200 MHz, DMSO-d6) δ: 3.21 (s, 3H, CH3), 3.63 (t, 2H, J = 4.9
Hz, CH2), 4.33 (t, 2H, J = 4.9 Hz, CH2), 6.41 (d, 1H, J = 2.0 Hz, H-3), 6.97-7.02 (m, 1H, H-6), 7.31
(d, 1H, J = 9.1 Hz, H-7), 7.43 (d, 1H, J = 2.0 Hz, H-2), 7.73 (m, 1H, H-4); 13C (50 MHz, DMSO-d6)
δ: 45.5 (t), 58.0 (q), 71.0 (t), 100.5 (d, JC3-F = 4.7 Hz), 104.8 (d, JC6-F = 23.0 Hz), 109.0 (d, JC4-F
26.1 Hz), 110.1 (d, JC7-F = 9.9 Hz), 128.2 (d, JC3a-F = 10.3 Hz), 130.9 (d), 131.6 (s), 157.0 (d, JC5-F
=
=
232.0 Hz). Anal. Calcd for C11H12FNO: C, 68.38; H, 6.26; N, 7.25. Found: C, 68.57; H, 6.06; N,
7.07. MS m/z 194 (MH+). HRMS: [MH]+, found 194.0966 C11H12FNO requires 194.0976.
5.1.1.5 1-(2-Methoxyethyl)-1H-pyrrolo[2,3-b]pyridine (20b)
1
Yellow oil; yield: 81%; H NMR (200 MHz, DMSO-d6) δ: 3.23 (s, 3H, CH3), 3.70 (t, 2H, J = 5.5
Hz, CH2), 4.43 (t, 2H, J = 5.5 Hz, CH2), 6.47 (d, 1H, J = 3.5 Hz, H-3), 7.09 (dd, 1H, J = 7.8, 4.7 Hz,
H-5), 7.55 (d, 1H, J = 3.5 Hz, H-2), 7.97 (dd, 1H, J = 4.7, 1.5 Hz, H-6), 8.26 (dd, 1H, J = 7.8, 1.5
Hz, H-4); 13C (50 MHz, DMSO-d6) δ: 43.3 (t), 57.9 (q), 70.6 (t), 98.8 (d), 115.5 (d), 120.0 (s), 128.4
(d), 129.4 (d), 142.2 (d), 147.0 (s). Anal. Calcd for C10H12N2O: C, 68.16; H, 6.86; N, 15.90. Found:
C, 67.99; H, 6.70; N, 15.64. MS m/z 177 (MH+). HRMS: [MH]+, found 177.1046 C10H12N2O
requires 177.1022.
5.1.2 General Procedure for the Synthesis of 1-(2-methoxyethyl)-1H-indole-3-carboxamides (13a-d)
To a solution of suitable indole 9a-d (1.2 mmol) in anhydrous acetonitrile (1.5 mL) chlorosulfonyl
isocyanate (2.4 mmol, 0.21 mL) was added dropwise at 0 °C. The reaction mixture was stirred at 0
°C for 30 min then a solution of acetone (8 mL) and water (1 mL) was added. The solution was
basified with 10% aqueous solution of potassium hydroxide and the obtained precipitate was
filtered off, dried and purified by column chromatography using dichloromethane/methanol (95/5)
(for compounds 13a,b) or dichloromethane/methanol (90/10) (for compounds 13c,d) as eluent.
5.1.2.1 1-(2-Methoxyethyl)-1H-indole-3-carboxamide (13a)
1
White solid; yield: 65%; mp: 147-148 °C; IR cm-1: 3373, 3184 (NH2), 1609 (CO); H NMR (200
MHz, DMSO-d6) δ: 3.23 (s, 3H, CH3), 3.67 (t, 2H, J = 5.2 Hz, CH2), 4.36 (t, 2H, J = 5.2 Hz, CH2),
6.88-7.24 (m, 4H, H-5, H-6 and NH2), 7.54 (dd, 1H, J = 6.8, 1.3 Hz, H-7), 8.04 (s, 1H, H-2), 8.15-
8.20 (m, 1H, H-4); 13C (50 MHz, DMSO-d6) δ: 45.8 (t), 58.1 (q), 70.5 (t), 109.6 (s), 110.3 (d), 120.5

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(d), 121.3 (d), 121.8 (d), 126.6 (s), 132.0 (d), 136.2 (s), 166.1 (s). Anal. Calcd for C12H14N2O2: C,
66.04; H, 6.47; N, 12.84. Found: C, 65.83; H, 6.32; N, 12.70. MS m/z 219 (MH+). HRMS: [MH]+,
found 219.1116 C12H14N2O2 requires 219.1128.
5.1.2.2 5-Methoxy-1-(2-methoxyethyl)-1H-indole-3-carboxamide (13b)
1
White solid; yield: 71%; mp: 120-121 °C; IR cm-1: 3380, 3182 (NH2), 1601 (CO); H NMR (200
MHz, DMSO-d6) δ: 3.22 (s, 3H, CH3), 3.65 (t, 2H, J = 5.0 Hz, CH2), 3.77 (s, 3H, CH3), 4.31 (t, 2H,
J = 5.0 Hz, CH2), 6.82 (dd, 1H, J = 8.9, 2.3 Hz, H-6), 7.27 (bs, 2H, NH2), 7.44 (d, 1H, J = 8.9 Hz,
13
H-7), 7.67 (d, 1H, J = 2.3 Hz, H-4), 7.98 (s, 1H, H-2); C (50 MHz, DMSO-d6) δ: 46.0 (t), 55.2
(q), 58.1 (q), 70.5 (t), 102.7 (d), 109.1 (s), 111.1 (d), 111.8 (d), 127.3 (s), 131.3 (s), 132.2 (d), 154.5
(s), 166.3 (s). Anal. Calcd for C13H16N2O3: C, 62.89; H, 6.50; N, 11.28. Found: C, 62.75; H, 6.40;
N, 11.04. MS m/z 249 (MH+). HRMS: [MH]+, found 249.1248 C13H16N2O3 requires 249.1234.
5.1.2.3 5-Bromo-1-(2-methoxyethyl)-1H-indole-3-carboxamide (13c)
White solid; yield: 93%; mp: 185 °C; IR cm-1: 3369, 3167 (NH2), 1609 (CO); 1H NMR (200 MHz,
DMSO-d6) δ: 3.22 (s, 3H, CH3), 3.65 (t, 2H, J = 5.0 Hz, CH2), 4.36 (t, 2H, J = 5.0 Hz, CH2), 6.95
(bs, 2H, NH2), 7.32 (dd, 1H, J = 8.7, 2.0 Hz, H-6), 7.56 (d, 1H, J = 8.7 Hz, H-7), 8.09 (s, 1H, H-2),
8.33 (d, 1H, J = 2.0 Hz, H-4); 13C (50 MHz, DMSO-d6) δ: 46.1 (t), 58.1 (q), 70.5 (t), 109.2 (s),
112.6 (d), 113.5 (s), 123.4 (d), 124.3 (d), 128.4 (s), 133.1 (d), 135.0 (s), 165.7 (s). Anal. Calcd for
C12H13BrN2O2: C, 48.50; H, 4.41; N, 9.43. Found: C, 48.36; H, 4.24; N, 9.67. MS m/z 297 (MH+).
HRMS: [MH]+, found 297.0219 C12H13BrN2O2 requires 297.0233.
5.1.2.4 5-Fluoro-1-(2-methoxyethyl)-1H-indole-3-carboxamide (13d)
White solid; yield: 98%; mp: 148 °C; IR cm-1: 3406, 3200 (NH2), 1606 (CO); 1H NMR (200 MHz,
DMSO-d6) δ: 3.22 (s, 3H, CH3), 3.66 (t, 2H, J = 5.2 Hz, CH2), 4.37 (t, 2H, J = 5.2 Hz, CH2), 6.92
(bs, 2H, NH2), 7.05 (td, 1H, J = 11.8, 9.2, 2.7 Hz, H-6), 7.59 (dd, 1H, J = 9.2, 4.5 Hz, H-7), 7.85
13
(dd, 1H, J = 11.8, 2.7, Hz, H-4), 8.11 (s, 1H, H-2); C (50 MHz, DMSO-d6) δ: 46.1 (t), 58.1 (q),
70.5 (t), 105.9.(d, JC6-F = 24.5 Hz), 110.0.(d, JC4-F = 26.2 Hz), 111.7(d, JC7-F = 9.9 Hz), 127.0 (s),
127.2 (s), 132.9 (s), 133.5 (d), 163.0 (d, JC5-F = 282.0 Hz), 165.9 (s). Anal. Calcd for C12H13FN2O2:
C, 61.01; H, 5.55; N, 11.86. Found: C, 61.25; H, 5.41; N, 11.67. MS m/z 237 (MH+). HRMS:
[MH]+, found 237.1051 C12H13FN2O2 requires 237.1034.
5.1.3 General Procedure for the Synthesis of 1-(2-methoxyethyl)-1H-indole-3-carbothioamides
(17a-d)

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To a solution of the appropriate carboxamide 13a-d (0.81 mmol) in THF (8 mL) Lawesson’s
reagent (0.41 mmol, 0.17 g) was added and the mixture was heated under reflux for 30 min. After
cooling the solvent was evaporated under reduce pressure and the residue purified by column
chromatography using petroleum ether/ethyl acetate (30/70) (for compounds 17a,b) or ethyl acetate
(for compounds 17c,d) as eluent.
5.1.3.1 1-(2-Methoxyethyl)-1H-indole-3-carbothioamide (17a)
1
Yellow solid; yield: 70%; mp: 151-152 °C; IR cm-1: 3377, 3182 (NH2), 1595 (CS); H NMR (200
MHz, DMSO-d6) δ: 3.23 (s, 3H, CH3), 3.68 (t, 2H, J = 5.2 Hz, CH2), 4.39 (t, 2H, J = 5.2 Hz, CH2),
7.15-7.26 (m, 2H, H-5 and H-6), 7.55-7.59 (m, 1H, H-7), 8.12 (s, 1H, H-2), 8.57-8.62 (m, 1H, H-4),
8.83 (bs, 1H, SH), 9.02 (bs, 1H, NH); 13C (50 MHz, DMSO-d6) δ: 45.8 (t), 58.1 (q), 70.4 (t), 110.6
(d), 115.5 (s), 121.0 (d), 121.8 (d), 122.1 (d), 126.0 (s), 132.0 (d), 136.7 (s), 193.0 (s). Anal. Calcd
for C12H14N2OS: C, 61.51; H, 6.02; N, 11.96. Found: C, 61.75; H, 6.16; N, 11.68. MS m/z 235
(MH+). HRMS: [MH]+, found 235.0923 C12H14N2OS requires 235.0900.
5.1.3.2 5-Methoxy-1-(2-methoxyethyl)-1H-indole-3-carbothioamide (17b)
1
Yellow solid; yield: 98%; mp: 133-134 °C; IR cm-1: 3371, 3160 (NH2), 1619 (CS); H NMR (200
MHz, DMSO-d6) δ: 3.22 (s, 3H, CH3), 3.66 (t, 2H, J = 5.1 Hz, CH2), 3.79 (s, 3H, CH3), 4.34 (t, 2H,
J = 5.1 Hz, CH2), 6.86 (dd, 1H, J = 8.9, 2.5 Hz, H-6), 7.47 (d, 1H, J = 8.9 Hz, H-7), 8.09 (s, 1H, H-
2), 8.19 (d, 1H, J = 2.5 Hz, H-4), 8.77 (bs, 1H, SH), 8.93 (bs, 1H, NH); 13C (50 MHz, DMSO-d6) δ:
46.0 (t), 55.3 (q), 58.1 (q), 70.4 (t), 103.9 (d), 111.4 (d), 111.8 (d), 115.0 (s), 126.8 (s), 131.8 (s),
132.4 (d), 154.9 (s), 192.9 (s). Anal. Calcd for C13H16N2O2S: C, 59.07; H, 6.10; N, 10.60. Found: C,
59.31; H, 5.99; N, 10.78. MS m/z 265 (MH+). HRMS: [MH]+, found 265.0994 C13H16N2O2S
requires 265.1005.
5.1.3.3 5-Bromo-1-(2-methoxyethyl)-1H-indole-3-carbothioamide (17c)
1
Yellow solid; yield: 92%; mp: 181-182 °C; IR cm-1: 3365, 3181 (NH2), 1629 (CS); H NMR (200
MHz, DMSO-d6) δ: 3.22 (s, 3H, CH3), 3.66 (t, 2H, J = 5.0 Hz, CH2), 4.38 (t, 2H, J = 5.0 Hz, CH2),
7.36 (dd, 1H, J = 8.8, 2.0 Hz, H-6), 7.58 (d, 1H, J = 8.8 Hz, H-7), 8.16 (s, 1H, H-2), 8.89 (d, 1H, J =
13
2.0 Hz, H-4), 8.96 (bs, 1H, SH), 9.10 (bs, 1H, NH); C (50 MHz, DMSO-d6) δ: 46.1 (t), 58.1 (q),
70.3 (t), 112.8 (d), 114.1 (s), 114.7 (s), 124.1 (d), 124.6 (d), 128.1 (s), 132.4 (d), 135.6 (s), 192.5 (s).
Anal. Calcd for C12H13BrN2OS: C, 46.02; H, 4.18; N, 8.94. Found: C, 45.88; H, 4.35; N, 8.78. MS
m/z 313 (MH+). HRMS: [MH]+, found 313.0021 C12H13BrN2OS requires 313.0005.

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5.1.3.4 5-Fluoro-1-(2-methoxyethyl)-1H-indole-3-carbothioamide (17d)
1
Yellow solid; yield: 99%; mp: 119-120 °C; IR cm-1: 3371, 3160 (NH2), 1647 (CS); H NMR (200
MHz, DMSO-d6) δ: 3.23 (s, 3H, CH3), 3.67 (t, 2H, J = 4.9 Hz, CH2), 4.39 (t, 2H, J = 4.9 Hz, CH2),
7.09 (td, 1H, J = 11.7, 9.1, 2.6 Hz, H-6), 7.62 (d, 1H, J = 9.1, 4.6 Hz, H-7), 8.20 (s, 1H, H-2), 8.44
13
(dd, 1H, J = 11.7, 2.6 Hz, H-4), 8.83 (s, 1H, SH), 9.02 (s, 1H, NH); C (50 MHz, DMSO-d6) δ:
46.2 (t), 58.1 (q), 70.3 (t) , 106.8 (d, JC6-F = 25.9 Hz), 110.2 (d, JC4-F = 26.3 Hz), 112.0 (d, JC7-F
=
10.0 Hz), 115.1 (d, JC7a-F = 4.7 Hz), 126.9 (d, JC3a-F = 11.0 Hz), 133.0 (d), 133.5 (s), 158.1 (d, JC5-F
= 233.0 Hz), 192.6 (s). Anal. Calcd for C12H13FN2OS: C, 57.12; H, 5.19; N, 11.10. Found: C, 56.94;
H, 5.43; N, 10.94. MS m/z 253 (MH+). HRMS: [MH]+, found 253.0817 C12H13FN2OS requires
253.0805.
5.1.4 Synthesis of 2-bromo-1-[1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]ethanone (21b)
Anhydrous AlCl3 (20.2 mmo,l 2.70 g) was slowly added to a solution of 1-(2-methoxyethyl)-1H-
pyrrolo[2,3-b]pyridine 20b (5.67 mmol, 1.0 g) in anhydrous DCM (15 mL). The reaction mixture
was heated under reflux and a solution of bromoacetyl bromide (5.67 mmol, 0.5 mL) in anhydrous
DCM (3 mL) was added dropwise. The resulting mixture was stirred under reflux for 40 min. After
cooling, water and ice were slowly added and the obtained precipitate was filtered off, dried and
purified by column chromatography using dichloromethane/ethyl acetate (85/15) as eluent. White
1
solid, yield: 70%; mp: 83-84 °C; IR cm-1: 1653 (CO); H NMR (200 MHz, DMSO-d6) δ: 3.25 (s,
3H, CH3), 3.79 (t, 2H, J = 5.2 Hz, CH2), 4.50 (t, 2H, J = 5.2 Hz, CH2), 4.68 (s, 2H, CH2), 7.34 (dd,
1H, J = 7.9, 4.7 Hz, H-5), 8.41 (dd, 1H, J = 4.7, 1.6 Hz, H-6), 8.48 (dd, 1H, J = 7.9, 1.6 Hz, H-4),
13
8.71 (s, 1H, H-2); C (50 MHz, DMSO-d6) δ: 33.0 (t), 44.4 (t), 58.0 (q), 69.7 (t), 111.1 (s), 118.2
(s), 118.8 (d), 129.9 (d), 138.2 (d), 144.3 (d), 147.8 (s), 186.3 (s). Anal. Calcd for C12H13BrN2O2: C,
48.50; H, 4.41; N, 9.43. Found: C, 48.34; H, 4.26; N, 9.59. MS m/z 297 (MH+). HRMS: [MH]+,
found 297.0207 C12H13BrN2O2 requires 297.0233.
5.1.5 General Procedure for the Synthesis of thiazoles (5a-q and 6a,b)
A suspension of the proper thioamides 16d, 17a-d, 18a-d and 19a-c (2 mmol) and bromoacetyl
derivatives 21a-c (2 mmol) in anhydrous ethanol (8 mL) was refluxed for 30 min. After cooling, the
precipitate obtained, was filtered off, dried, and recrystallized from ethanol to give the desidered
thiazoles 5a-q and 6a,b.
5.1.5.1 3-{2-[1-(2-Methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1H-pyrrolo[2,3-b]pyridine (5a)

ACCEPTED MANUSCRIPT
Yellow solid; yield: 66%; mp: 238-239 °C; IR cm-1: 3300 (NH); 1H NMR (200 MHz, DMSO-d6) δ:
3.25 (s, 3H, CH3), 3.74 (t, 2H, J = 5.1 Hz, CH2), 4.48 (t, 2H, J = 5.1 Hz, CH2), 7.26-7.35 (m, 2H, H-
5’ and H-6’), 7.49 (dd, 1H, J = 7.9, 5.3 Hz, H-5’’), 7.64-7.69 (m, 1H, H-7’), 7.87 (s, 1H, H-5), 8.24-
8.33 (m, 3H, H-2’, H-2’’ and H-4’), 8.48 (dd, 1H, J = 5.3, 1.2 Hz, H-6’’), 9.00 (d, 1H, J = 7.9 Hz,
H-4’’), 12.60 (bs, 1H, NH); 13C (50 MHz, DMSO-d6) δ: 45.8 (t), 58.1 (q), 70.7 (t), 108.9 (d), 109.5
(s), 111.0 (d), 111.3 (s), 116.2 (d), 120.3 (d), 121.2 (d), 121.5 (s), 122.5 (d), 124.6 (s), 126.9 (d),
130.3 (d), 135.2 (d), 135.6 (s), 136.6 (s), 136.9 (d), 147.8 (s), 162.3 (s). Anal. Calcd for
C21H18N4OS: C, 67.36; H, 4.85; N, 14.96. Found: C, 67.24; H, 4.69; N, 14.79. MS m/z 375 (MH+).
HRMS: [MH]+, found 375.1260 C21H18N4OS requires 375.1274.
5.1.5.2
3-{2-[1-(2-Methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-methyl-1H-pyrrolo[2,3-
b]pyridine (5b)
1
Orange solid; yield: 70%; mp: 248-249 °C; H NMR (200 MHz, DMSO-d6) δ: 3.24 (s, 3H, CH3),
3.73 (t, 2H, J = 5.0 Hz, CH2), 4.00 (s, 3H, CH3), 4.47 (t, 2H, J = 5.0 Hz, CH2), 7.28-7.32 (m, 2H, H-
5’ and H-6’), 7.47 (dd, 1H, J = 7.9, 5.2 Hz, H-5’’), 7.63-7.67 (m, 1H, H-7’), 7.82 (s, 1H, H-5), 8.25-
8.33 (m, 3H, H-2’, H-2’’ and H-4’), 8.50 (d, 1H, J = 5.2 Hz, H-6’’), 8.90 (d, 1H, J = 7.9 Hz, H-4’’);
13C (50 MHz, DMSO-d6) δ: 31.6 (q), 45.8 (t), 58.1 (q), 70.6 (t), 109.3 (s), 109.6 (s), 110.9 (s), 111.1
(d), 116.1 (d), 120.0 (d), 120.4 (s), 121.1 (d), 122.5 (d), 124.6 (s), 129.7 (d), 130.4 (s), 130.5 (d),
130.6 (d), 131.5 (d), 136.6 (s), 141.8 (d), 162.1 (s). Anal. Calcd for C22H20N4OS: C, 68.02; H, 5.19;
N, 14.42. Found: C, 68.17; H, 5.39; N, 14.30. MS m/z 389 (MH+). HRMS: [MH]+, found 389.1450
C22H20N4OS requires 389.1431.
5.1.5.3
1-(2-Methoxyethyl)-3-{2-[1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1H-
pyrrolo[2,3-b]pyridine (5c)
1
Orange solid; yield: 91%; mp: 219-220 °C; H NMR (200 MHz, DMSO-d6) δ: 3.24 (s, 3H, CH3),
3.26 (s, 3H, CH3), 3.71-3.82 (m, 4H, 2 x CH2), 4.48 (t, 2H, J = 5.0 Hz, CH2), 4.59 (t, 2H, J = 5.0
Hz, CH2), 7.26-7.35 (m, 2H, H-5’ and H-6’), 7.42 (dd, 1H, J = 7.9, 5.0 Hz, H-5’’), 7.62-7.70 (m,
1H, H-7’), 7.82 (s, 1H, H-5), 8.24-8.31 (m, 3H, H-2’, H-2’’ and H-4’), 8.46 (d, 1H, J = 5.0, 1.2 Hz,
H-6’’), 8.83 (dd, 1H, J = 7.9, 1.2 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ: 44.4 (t), 45.8 (t), 58.1 (2 x
q), 70.5 (t), 70.7 (t), 107.9 (d), 109.4 (s), 109.5 (s), 111.0 (d), 116.4 (d), 119.0 (s), 120.4 (d), 121.1
(d), 122.4 (d), 124.6 (s), 129.2 (d), 130.3 (d), 131.5 (d), 136.6 (s), 140.4 (d), 144.8 (s), 148.1 (s),
162.1 (s). Anal. Calcd for C24H24N4O2S: C, 66.64; H, 5.59; N, 12.95. Found: C, 66.80; H, 5.44; N,
12.74. MS m/z 433 (MH+). HRMS: [MH]+, found 433.1675 C24H24N4O2S requires 433.1693.

ACCEPTED MANUSCRIPT
5.1.5.4
1-(2-Methoxyethyl)-3-[2-(1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-
b]pyridine (5d)
1
Yellow solid; yield: 98%; mp: 242-243 °C; H NMR (200 MHz, DMSO-d6) δ: 3.14 (s, 3H, CH3),
3.79 (t, 2H, J = 5.3 Hz, CH2), 3.92 (s, 3H, CH3), 4.56 (t, 2H, J = 5.3 Hz, CH2), 7.30-7.37 (m, 3H, H-
5’, H-5’’ and H-6’), 7.56-7.64 (m, 1H, H-7’), 7.76 (s, 1H, H-5), 8.23 (s, 1H, H-2’), 8.25 (s, 1H, H-
2’’), 8.30-8.42 (m, 2H, H-4’ and H-6’’), 8.73 (dd, 1H, J = 8.0, 1.5 Hz, H-4’’); 13C (50 MHz,
DMSO-d6) δ: 32.9 (q), 44.4 (t), 58.1 (q), 70.5 (t), 107.8 (d), 109.2 (s), 109.5 (s), 110.7 (d), 116.3
(d), 118.9 (s), 120.4 (d), 121.1 (d), 122.5 (d), 124.5 (s), 129.1 (d), 130.8 (d), 131.3 (d), 137.1 (s),
140.5 (d), 144.9 (s), 148.2 (s), 162.1 (s). Anal. Calcd for C22H20N4OS: C, 68.02; H, 5.19; N, 14.42.
C, 68.17; H, 5.00; N, 14.31. MS m/z 389 (MH+). HRMS: [MH]+, found 389.1416 C22H20N4OS
requires 389.1431.
5.1.5.5 3-{2-[5-Methoxy-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1H-pyrrolo[2,3-
b]pyridine (5e)
1
Yellow solid; yield: 73%; mp: 237-238 °C; IR cm-1: 3299 (NH); H NMR (200 MHz, DMSO-d6) δ:
3.24 (s, 3H, CH3), 3.72 (t, 2H, J = 5.0 Hz, CH2), 3.90 (s, 3H, CH3), 4.43 (t, 2H, J = 5.0 Hz, CH2),
6.94 (dd, 1H, J = 8.9, 2.4 Hz, H-6’), 7.44 (dd, 1H, J = 7.9, 5.2 Hz, H-5’’), 7.57 (d, 1H, J = 8.9 Hz,
H-7’), 7.82 (s, 1H, H-5), 7.88 (d, 1H, J = 2.4 Hz, H-4’), 8.17 (s, 1H, H-2’), 8.26 (d, 1H, J = 2.2 Hz,
H-2’’), 8.47 (dd, 1H, J = 5.2, 1.2 Hz, H-6’’), 9.04 (d, 1H, J = 7.9 Hz, H-4’’), 12.54 (bs, 1H, NH);
13C (50 MHz, DMSO-d6) δ: 45.9 (t), 55.2 (q), 58.1 (q), 70.7 (t), 101.9 (d), 108.2 (d), 109.2 (s),
111.1 (s), 111.8 (d), 112.5 (d), 116.0 (d), 120.7 (s), 125.1 (s), 126.4 (d), 130.5 (d), 131.6 (s), 134.3
(d), 137.8 (d), 142.5 (s), 148.0 (s), 154.9 (s), 162.5 (s). Anal. Calcd for C22H20N4O2S: C, 65.33; H,
4.98; N, 13.85. Found: C, 65.55; H, 4.82; N, 13.70. MS m/z 405 (MH+). HRMS: [MH]+, found
405.1408 C22H20N4O2S requires 405.1380.
5.1.5.6
3-{2-[5-Methoxy-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-methyl-1H-
pyrrolo[2,3-b]pyridine (5f)
1
Yellow solid; yield: 70%; mp: 223-224 °C; H NMR (200 MHz, DMSO-d6) δ: 3.24 (s, 3H, CH3),
3.71 (t, 2H, J = 5.1 Hz, CH2), 3.89 (s, 3H, CH3), 3.98 (s, 3H, CH3), 4.42 (t, 2H, J = 5.1 Hz, CH2),
6.94 (dd, 1H, J = 8.9, 2.5 Hz, H-6’), 7.42 (dd, 1H, J = 7.9, 5.0 Hz, H-5’’), 7.56 (d, 1H, J = 8.9 Hz,
H-7’), 7.76 (s, 1H, H-5), 7.82 (d, 1H, J = 2.5 Hz, H-4’), 8.18 (s, 1H, H-2’), 8.27 (s, 1H, H-2’’), 8.49
(dd, 1H, J = 5.0, 1.2 Hz, H-6’’), 8.94 (dd, 1H, J = 7.9, 1.2 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ:
32.1 (q), 46.0 (t), 55.3 (q), 58.1 (q), 70.7 (t), 102.1 (d), 107.7 (d), 109.0 (s), 109.5 (s), 111.8 (d),

ACCEPTED MANUSCRIPT
112.4 (d), 116.0 (d), 116.1 (s), 122.1 (d), 124.9 (s), 125.1 (s), 129.7 (d), 130.6 (d), 131.7 (s), 132.3
(d), 154.9 (s), 155.1 (s), 162.4 (s). Anal. Calcd for C23H22N4O2S: C, 66.01; H, 5.30; N, 13.39.
Found: C, 66.14; H, 5.05; N, 13.23. MS m/z 419 (MH+). HRMS: [MH]+, found 419.1552
C23H22N4O2S requires 419.1536.
5.1.5.7 1-(2-Methoxyethyl)-3-{2-[5-methoxy-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-
1H-pyrrolo[2,3-b]pyridine (5g)
1
Yellow solid; yield: 60%; mp: 214-215 °C; H NMR (200 MHz, DMSO-d6) δ: 3.25 (s, 3H, CH3),
3.27 (s, 3H, CH3), 3.69-3.81 (m, 4H, 2 x CH2), 3.91 (s, 3H, CH3), 4.43 (t, 2H, J = 5.1 Hz, CH2),
4.55 (t, 2H, J = 5.1 Hz, CH2), 6.95 (dd, 1H, J = 8.9, 2.5 Hz, H-6’), 7.33 (dd, 1H, J = 7.9, 4.9 Hz, H-
5’’), 7.57 (d, 1H, J = 8.9 Hz, H-7’), 7.74 (s, 1H, H-5), 7.88 (d, 1H, J = 2.5 Hz, H-4’), 8.15 (s, 1H,
H-2’), 8.23 (s, 1H, H-2’’), 8.41 (dd, 1H, J = 4.9, 1.4, Hz H-6’’), 8.81 (dd, 1H, J = 7.9, 1.4 Hz, H-
4’’); 13C (50 MHz, DMSO-d6) δ: 44.2 (t), 46.0 (t), 55.1 (q), 58.1 (2 x q), 70.5 (t), 70.7 (t), 102.0 (d),
107.8 (s), 108.9 (s), 109.5 (s), 111.9 (d), 112.6 (d), 116.2 (d), 116.8 (d), 119.6 (s), 125.1 (s), 129.1
(d), 130.6 (d), 131.6 (d), 141.6 (d), 144.1 (s), 147.7 (s), 155.0 (s), 162.5 (s). Anal. Calcd for
C25H26N4O3S: C, 64.91; H, 5.67; N, 12.11. Found: C, 64.78; H, 5.48; N, 12.01. MS m/z 463 (MH+).
HRMS: [MH]+, found 463.1783 C25H26N4O3S requires 463.1798.
5.1.5.8
1-(2-Methoxyethyl)-3-[2-(5-methoxy-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-
pyrrolo[2,3-b]pyridine (5h)
1
Orange solid; yield: 96%; mp: 238-239 °C; H NMR (200 MHz, DMSO-d6) δ: 3.27 (s, 3H, CH3),
3.78 (t, 2H, J = 5.1 Hz, CH2), 3.88 (s, 3H, CH3), 3.91 (s, 3H, CH3), 4.55 (t, 2H, J = 5.1 Hz, CH2),
6.96 (dd, 1H, J = 8.9, 2.5 Hz, H-6’), 7.33 (dd, 1H, J = 7.9, 4.9 Hz, H-5’’), 7.50 (d, 1H, J = 8.9 Hz,
H-7’), 7.72 (s, 1H, H-5), 7.90 (d, 1H, J = 2.5 Hz, H-4’), 8.16 (s, 1H, H-2’), 8.22 (s, 1H, H-2’’), 8.42
(dd, 1H, J = 4.9, 1.4 Hz, H-6’’), 8.81 (dd, 1H, J = 7.9, 1.4 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ:
33.05 (q), 44.1 (t), 55.2 (q), 58.0 (q), 70.4 (t), 101.9 (d), 107.4 (d), 108.8 (s), 109.5 (s), 110.6 (d),
111.6 (d), 112.5 (d), 116.2 (d), 119.0 (s), 125.1 (s), 131.0 (d), 131.2 (s), 131.5 (d), 140.5 (d), 144.8
(s), 148.1 (s), 155.0 (s), 162.4 (s). Anal. Calcd for C23H22N4O2S: C, 66.01; H, 5.30; N, 13.39.
Found: C, 65.75; H, 5.06; N, 13.25. MS m/z 419 (MH+). HRMS: [MH]+, found 419.1549
C23H22N4O2S requires 419.1536.
5.1.5.9
3-{2-[5-Bromo-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1H-pyrrolo[2,3-
b]pyridine (5i)

ACCEPTED MANUSCRIPT
Orange solid; yield: 75%; mp: 232 °C; IR cm-1: 3355 (NH) ;1H NMR (200 MHz, DMSO-d6) δ: 3.24
(s, 3H, CH3), 3.73 (t, 2H, J = 5.0 Hz, CH2), 4.48 (t, 2H, J = 5.0 Hz, CH2), 7.38-7.46 (m, 2H, H-5’’
and H-6’), 7.67 (d, 1H, J = 8.8 Hz, H-7’), 7.84 (s, 1H, H-5), 8.21 (d, 1H, J = 2.3 Hz, H-2’’), 8.28 (s,
1H, H-2’), 8.45 (dd, 1H, J = 5.1, 1.2 Hz, H-6’’), 8.52 (d, 1H, J = 1.8, Hz, H-4’),8.89 (d, 1H, J = 7.9
13
Hz, H-4’’), 12.42 (bs, 1H, NH); C (50 MHz, DMSO-d6) δ: 46.0 (t), 58.1 (q), 70.7 (t), 109.2 (d),
111.2 (s), 113.1 (s), 113.2 (d), 113.7 (s), 116.1 (d), 120.8 (s), 122.6 (d), 123.7 (s), 124.9 (d), 126.2
(s), 126.6 (d), 131.4 (d), 134.6 (d), 135.4 (s), 137.4 (d), 148.2 (s), 161.6 (s). Anal. Calcd for
C21H17BrN4OS: C, 55.64; H, 3.78; N, 12.36. Found: C, 55.43; H, 3.68; N, 12.51. MS m/z 453
(MH+). HRMS: [MH]+, found 453.0395 C21H17BrN4OS requires 453.0379.
5.1.5.10
3-{2-[5-Bromo-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-methyl-1H-
pyrrolo[2,3-b]pyridine (5j)
1
Orange solid; yield: 84%; mp: 250-251 °C; H NMR (200 MHz, DMSO-d6) δ: 3.24 (s, 3H, CH3),
3.72 (t, 2H, J = 5.0 Hz, CH2), 3.96 (s, 3H, CH3), 4.47 (t, 2H, J = 5.0 Hz, CH2), 7.34 (dd, 1H, J =
7.9, 4.9 Hz, H-5’’), 7.44 (dd, 1H, J = 8.8, 1.9 Hz, H-6’), 7.67 (d, 1H, J = 8.8 Hz, H-7’), 7.77 (s, 1H,
H-5), 8.21-8.26 (m, 2H, H-2’ and H-2’’), 8.44 (dd, 1H, J = 4.9, 1.3 Hz, H-6’’), 8.50 (d, 1H, J = 1.9,
Hz, H-4’),8.75 (dd, 1H, J = 7.9, 1.3 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ: 31.9 (q), 46.0 (t), 58.1
(q), 70.7 (t), 108.1 (d), 109.2 (s), 109.5 (s), 113.2 (d), 113.7 (s), 116.0 (d), 122.6 (d), 125.0 (d),
126.2 (s), 129.5 (d), 131.4 (d), 131.6 (d), 135.4 (s), 140.1 (d), 140.3 (s), 145.2 (s), 148.6 (s), 161.4
(s). Anal. Calcd for C22H19BrN4OS: C, 56.54; H, 4.10; N, 11.99. Found: C, 56.30; H, 3.95; N,
11.89. MS m/z 467 (MH+). HRMS: [MH]+, found 467.0525 C22H19BrN4OS requires 467.0536.
5.1.5.11
3-{2-[5-Bromo-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-(2-methoxyethyl)-
1H-pyrrolo[2,3-b]pyridine (5k)
1
Orange solid; yield: 64%; mp: 149-150 °C; H NMR (200 MHz, DMSO-d6) δ: 3.24 (s, 3H, CH3),
3.30 (s, 3H, CH3), 3.70-3.72 (m, 4H, 2 x CH2), 4.45-4.58 (m, 4H, 2 x CH2), 7.31 (dd, 1H, J = 7.9,
4.8 Hz, H-5’’), 7.44 (dd, 1H, J = 8.8, 1.9 Hz, H-6’), 7.67 (d, 1H, J = 8.8 Hz, H-7’), 7.77 (s, 1H, H-
5), 8.20-8.26 (m, 2H, H-2’ and H-2’’), 8.39 (dd, 1H, J = 4.8, 1.2 Hz, H-6’’), 8.52 (d, 1H, J = 1.9,
Hz, H-4’), 8.69 (dd, 1H, J = 7.9, 1.2 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ: 44.2 (t), 46.0 (t), 58.1
(2 x q). 70.5 (t), 70.7 (t), 108.0 (d), 109.2 (s), 109.5 (s), 113.2 (d), 113.7 (s), 116.3 (d), 118.7 (s),
122.7 (d), 125.0 (d), 126.2 (s), 128.8 (d), 130.9 (s), 131.0 (d), 131.4 (d), 135.4 (s), 140.8 (d), 148.7
(s), 161.4 (s). Anal. Calcd for C24H23BrN4O2S: C, 56.36; H, 4.53; N, 10.95. Found: C, 56.21; H,
4.27; N, 10.81. MS m/z 511 (MH+). HRMS: [MH]+, found 511.0811 C24H23BrN4O2S requires
511.0798.

ACCEPTED MANUSCRIPT
5.1.5.12
3-[2-(5-Bromo-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-(2-methoxyethyl)-1H-
pyrrolo[2,3-b]pyridine (5l)
Yellow solid; yield: 84%; mp: 240-241 °C; 1H NMR (200 MHz, DMSO-d6) δ: 3.29 (s, 3H, CH3),
3.79 (t, 2H, J = 5.1 Hz, CH2), 3.89 (s, 3H, CH3), 4.57 (t, 2H, J = 5.1 Hz, CH2), 7.36-7.45 (m, 2H, H-
5’’ and H-6’), 7.56 (d, 1H, J = 8.7 Hz, H-7’), 7.78 (s, 1H, H-5), 8.24-8.26 (m, 2H, H-2’ and H-2’’),
8.45-8.47 (m, 2H, H-4’ and H-6’’), 8.82 (dd, 1H, J = 7.9, 1.3 Hz, H-4’’); 13C (50 MHz, DMSO-d6)
δ: 33.1 (q), 44.4 (t), 58.1 (q), 70.5 (t), 108.1 (d), 108.9 (s), 109.7 (s), 112.8 (d), 113.8 (s), 116.2 (d),
119.1 (s), 122.7 (d), 124.9 (d), 126.1 (s), 128.9 (d), 131.5 (d), 132.0 (d), 135.8 (s), 140.2 (d), 144.6
(s), 148.4 (s), 161.5 (s). Anal. Calcd for C22H19BrN4OS: C, 56.54; H, 4.10; N, 11.99. Found: C,
56.75; H, 3.95; N, 11.85. MS m/z 467 (MH+). HRMS: [MH]+, found 467.0552 C22H19BrN4OS
requires 467.0536.
5.1.5.13 3-{2-[5-Fluoro-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1H-pyrrolo[2,3-
b]pyridine (5m)
Orange solid; yield: 70%; mp: 203-204 °C; IR cm-1: 3237 (NH); 1H NMR (200 MHz, DMSO-d6) δ:
3.25 (s, 3H, CH3), 3.73 (t, 2H, J = 5.0 Hz, CH2), 4.48 (t, 2H, J = 5.0 Hz, CH2), 7.17 (td, 1H, J =
11.7, 9.2, 2.5 Hz, H-6’), 7.46 (dd, 1H, J = 7.9, 5.2 Hz, H-5’’), 7.70 (dd, 1H, J = 9.2, 4.5 Hz, H-7’),
7.85 (s, 1H, H-5), 8.03 (dd, 1H, J = 11.7, 2.5 Hz, H-4’), 8.26 (d, 1H, J = 2.3 Hz, H-2’’), 8.30 (s, 1H,
H-2’), 8.47 (dd, 1H, J = 5.2, 1.2 Hz, H-6’’), 8.92 (d, 1H, J = 7.9 Hz, H-4’’), 12.53 (bs, 1H, NH); 13C
(50 MHz, DMSO-d6) δ: 46.1 (t), 58.1 (q), 70.7 (t), 105.2 (d, JC6’-F = 25.5 Hz), 109.0 (d), 109.6 (d,
JC7’a-F = 4.9 Hz), 110.7 (d, JC4’-F = 25.8 Hz), 111.3 (s), 112.4 (d, JC7’-F = 9.6 Hz), 116.2 (d), 121.0
(s), 124.8 ( d, JC3’a-F = 10.8 Hz), 126.9 (d), 131.9 (d), 133.3 (s), 134.8 (d), 137.1 (d), 141.7 (s), 148.0
(s), 158.1 (d, JC5’-F = 234 Hz), 161.9 (s). Anal. Calcd for C21H17FN4OS: C, 64.27; H, 4.37; N, 14.28.
Found: C, 64.01; H, 4.26; N, 14.57. MS m/z 393 (MH+). HRMS: [MH]+, found 393.1199
C21H17FN4OS requires 393.1180.
5.1.5.14
3-{2-[5-Fluoro-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-methyl-1H-
pyrrolo[2,3-b]pyridine (5n)
1
Yellow solid; yield: 75%; mp: 252-253 °C; H NMR (200 MHz, DMSO-d6) δ: 3.25 (s, 3H, CH3),
3.72 (t, 2H, J = 5.1 Hz, CH2), 3.97 (s, 3H, CH3), 4.47 (t, 2H, J = 5.1 Hz, CH2), 7.17 (td, 1H, J =
11.7, 9.2, 2.5, Hz, H-6’), 7.37 (dd, 1H, J = 7.9, 4.9 Hz, H-5’’), 7.69 (dd, 1H, J = 9.2, 4.5 Hz, H-7’),
7.77 (s, 1H, H-5), 8.07(dd, 1H, J = 11.7, 2.5 Hz, H-4’), 8.27 (bs, 2H, H-2’ and H-2’’), 8.44 (dd, 1H,
J = 4.9, 1.3 Hz, H-6’’), 8.74 (dd, 1H, J = 7.9, 1.3 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ: 31.7 (q),
46.0 (t), 58.1 (q), 70.7 (t), 105.3 (d, JC6’-F = 24.9 Hz), 107.5 (s), 107.6 (d), 109.3 (s), 109.7 (s), 110.7

ACCEPTED MANUSCRIPT
(d, JC4’-F = 26.2 Hz), 112.3 (d, JC7’-F = 10.2 Hz) 116.1 (d), 116.3 (s), 124.8 (d, JC3’a-F = 10.9 Hz),
129.7 (d), 130.9 (d), 131.8 (d), 133.3 (s), 140.5 (d), 148.7 (s), 158.2 (d, JC5’-F = 235 Hz), 161.6 (s).
Anal. Calcd for C22H19FN4OS C, 65.01; H, 4.71; N, 13.78. Found: C, 65.22; H, 4.56; N, 14.05. MS
m/z 407 (MH+). HRMS: [MH]+, found 407.1324 C22H19FN4OS requires 407.1336.
5.1.5.15 3-{2-[5-Fluoro-1-(2-methoxyethyl)-1H-indol-3-yl]-1,3-thiazol-4-yl}-1-(2-methoxyethyl)-
1H-pyrrolo[2,3-b]pyridine (5o)
Orange solid; yield: 72%; mp: 173°C; 1H NMR (200 MHz, DMSO-d6) δ: 3.25 (s, 3H, CH3), 3.28 (s,
3H, CH3), 3.71-3.82 (m, 4H, 2 x CH2), 4.45-4.59 (m, 4H, 2 x CH2), 7.17 (td, 1H, J = 11.7, 9.2, 2.5
Hz, H-6’), 7.32 (dd, 1H, J = 7.9, 4.8 Hz, H-5’’), 7.70 (dd, 1H, J = 9.2, 4.5 Hz, H-7’), 7.76 (s, 1H, H-
5), 8.06 (dd, 1H, J = 11.7, 2.5 Hz, H-4’), 8.25 (s, 1H, ArH), 8.27 (s, 1H, ArH), 8.40 (dd, 1H, J =
4.8, 1.4 Hz, H-6’’), 8.67 (dd, 1H, J = 7.9, 1.4 Hz, H-4’’); 13C (50 MHz, DMSO-d6) δ: 44.1 (t), 46.0
(t), 58.0 (q), 58.1 (q), 70.5 (t), 70.7 (t), 105.4 (d, JC6’-F = 24.1 Hz), 107.6 (d), 109.4 (s), 109.7 (d,
JC7’a-F = 4.2 Hz), 110.6 (d, JC4’-F = 26.4 Hz), 112.4 (d, JC7’-F = 10.0Hz), 116.3 (d), 118.4. (s), 125.0 (
d, JC3’a-F = 10.7 Hz), 128.9 (d), 130.3 (d), 131.8 (d), 133.3 (s), 141.3 (d), 145.7 (s), 148.7 (s), 158.2
(d, JC5’-F = 234 Hz), 161.6 (s). Anal. Calcd for C24H23FN4O2S: C, 63.98; H, 5.15; N, 12.44. Found:
C, 64.22; H, 5.02; N, 12.30. MS m/z 451 (MH+). HRMS: [MH]+, found 451.1623 C24H23FN4O2S
requires 451.1599.
5.1.5.16 3-[2-(5-Fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-(2-methoxyethyl)-1H-
pyrrolo[2,3-b]pyridine (5p)
1
Yellow solid; yield: 89%; mp: 209-210°C; H NMR (200 MHz, DMSO-d6) δ: 3.28 (s, 3H, CH3),
3.79 (t, 2H, J = 5.3 Hz, CH2), 3.92 (s, 3H, CH3), 4.56 (t, 2H, J = 5.3 Hz, CH2), 7.19 (td, 1H, J =
11.6, 9.2, 2.5 Hz, H-6’), 7.33 (dd, 1H, J = 7.9, 4.8 Hz, H-5’’), 7.63 (dd, 1H, J = 9.2, 4.4 Hz, H-7’),
7.76 (s, 1H, H-5), 8.07 (dd, 1H, J = 11.6, 2.5 Hz, H-4’), 8.25 (s, 1H, ArH), 8.29 (s, 1H, ArH), 8.40
13
(dd, 1H, J = 4.8, 1.4 Hz, H-6’’), 8.69 (d, 1H, J = 7.9, 1.4 Hz, H-4’’); C (50 MHz, DMSO-d6) δ:
33.2 (q), 44.4 (t), 58.1 (q), 70.5 (t), 105.4 (d, JC6’-F = 24.4 Hz), 107.9 (d), 109.4 (d, JC7’a-F = 4.6 Hz),
109.6 (s), 110.7 (d, JC4’-F = 26.0 Hz), 112.1 (d, JC7’-F = 9.9 Hz), 116.3 (d), 119.0 (s), 124.8 ( d, JC3’a-F
= 10.8 Hz), 129.2 (d), 131.4 (d), 132.4 (d), 133.8 (s), 140.3 (d), 144.7 (s), 148.3 (s), 158.2 (d, JC5’-F
= 234 Hz), 161.8 (s). Anal. Calcd for C22H19FN4OS: C, 65.01; H, 4.71; N, 13.78. Found: C, 64.77;
H, 4.57; N, 13.52. MS m/z 407 (MH+). HRMS: [MH]+, found 407.1312 C22H19FN4OS requires
407.1336.

ACCEPTED MANUSCRIPT
5.1.5.17 3-[2-(5-Fluoro-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-(2-methoxyethyl)-1H-pyrrolo[2,3-
b]pyridine (5q)
1
Green solid; yield: 56%; mp: 185-186°C; IR cm-1: 3454 (NH); H NMR (200 MHz, DMSO-d6) δ:
3.27 (s, 3H, CH3), 3.79 (t, 2H, J = 5.1 Hz, CH2), 4.59 (t, 2H, J = 5.1 Hz, CH2), 7.12 (td, 1H, J =
11.6, 9.2, 2.5 Hz, H-6’), 7.39-7.46 (m, 1H, H-7’), 7.55 (dd, 1H, J = 8.9, 4.6 Hz, H-5’’), 7.82 (s, 1H,
H-5), 8.04 (dd, 1H, J = 11.6, 2.5 Hz, H-4’), 8.29 (d, 1H, J = 2.8 Hz, H-2’), 8.32 (s, 1H, H-2’’), 8.47
(d, 1H, J = 4.6 Hz, H-6’’), 8.83 (d, 1H, J = 8.9 Hz, H-4’’), 11.98 (bs, 1H, NH); 13C (50 MHz,
DMSO-d6) δ: 44.3 (t), 58.1 (q), 70.5 (t), 105.2 (d, JC6’-F = 24.0 Hz), 107.9 (d), 109.6 (s), 110.4 (s),
110.7 (d, JC4’-F = 26.3 Hz), 113.5 (d, JC7’-F = 10.0Hz), 116.3 (d), 119.0. (s), 124.5 (d, JC3’a-F = 10.8
Hz), 128.8 (d), 129.2 (d), 131.4 (d), 133.2 (s), 140.4 (d), 144.7 (s), 148.2 (s), 158.0 (d, JC5’-F = 233
Hz), 162.2 (s). Anal. Calcd for C21H17FN4OS: C, 64.27; H, 4.37; N, 14.28. Found: C, 64.17; H,
4.62; N, 14.12. MS m/z 393 (MH+). HRMS: [MH]+, found 393.1197 C21H17FN4OS requires
393.1180.
5.1.5.18 Tert-butyl 3-{4-[1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1,3-thiazol-2-yl}-1H-
indole-1-carboxylate (6a)
Yellow solid; yield: 94%; mp: 179-180 °C; IR cm-1: 1738 (CO); H NMR (200 MHz, DMSO-d6) δ:
1
1.68 (s, 9H, 3 x CH3), 3.27 (s, 3H, CH3), 3.79 (t, 2H, J = 5.2 Hz, CH2), 4.58 (t, 2H, J = 5.2 Hz,
CH2), 7.36-7.50 (m, 3H, H-5’, H-5’’ and H-6’), 7.97 (s, 1H, H-5), 8.15-8.20 (m, 1H, H-7’), 8.32-
8.49 (m, 4H, H-2’, H-2’’, H-4’ and H-6’’), 8.76 (dd, 1H, J = 8.0, 1.3 Hz, H-4’’); 13C (50 MHz,
DMSO-d6) δ: 27.6 (3 x q), 44.3 (t), 58.1 (q), 70.5 (t), 84.9 (s), 99.6 (s), 109.3 (s), 110.0 (d), 115.0
(d), 116.4 (d), 118.6 (s), 121.3 (d), 123.9 (d), 125.5 (d), 125.6 (d), 126.6 (s), 129.2 (d), 131.0 (d),
135.0 (s), 140.7 (d), 145.1 (s), 148.6 (s), 149.3 (s), 159.8 (s). Anal. Calcd for C26H26N4O3S: C,
65.80; H, 5.52; N, 11.81. Found: C, 65.69; H, 5.28; N, 11.62. MS m/z 475 (MH+). HRMS: [MH]+,
found 475.1776 C26H26N4O3S requires 475.1798.
5.1.5.19 Tert-butyl 5-bromo-3-{4-[1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-1,3-thiazol-
2-yl}-1H-indole-1-carboxylate (6b)
Yellow solid; yield: 71%; mp: 157-158 °C; IR cm-1: 1742 (CO); 1H NMR (200 MHz, DMSO-d6) δ:
1.69 (s, 9H, 3 x CH3), 3.30 (s, 3H, CH3), 3.79 (t, 2H, J = 5.1 Hz, CH2), 4.55 (t, 2H, J = 5.1 Hz,
CH2), 7.29 (dd, 1H, J = 7.9, 4.7 Hz, H-5’’), 7.65 (dd, 1H, J = 8.9, 2.0 Hz, H-6’), 7.94 (s, 1H, H-5),
8.10 (d, 1H, J = 8.9 Hz, H-7’), 8.21 (s, 1H, H-2’’), 8.37-8.39 (m, 2H, H-2’ and H-6’’), 8.63 (dd, 1H,
13
J = 7.9, 1.5 Hz, H-4’’), 8.68 (d, 1H, J = 2.0 Hz, H-4’); C (50 MHz, DMSO-d6) δ: 27.6 (3 x q),
43.9 (t), 58.0 (q), 70.7 (t), 85.3 (s), 99.5 (s), 108.9 (s), 109.8 (d), 114.1 (s), 116.3 (d), 116.8 (d),

ACCEPTED MANUSCRIPT
118.0 (s), 123.8 (d), 126.7 (d), 127.9 (d), 128.4 (s), 128.6 (d), 129.7 (d), 133.8 (s), 141.8 (d), 146.2
(s), 148.3 (s), 149.6 (s), 159.3 (s). Anal. Calcd for C26H25BrN4O3S: C, 56.42; H, 4.55; N, 10.12.
Found: C, 56.19; H, 4.44; N, 9.89. MS m/z 553 (MH+). HRMS: [MH]+, found 553.0892
C26H25BrN4O3S requires 553.0903.
5.1.6 General Procedure for the Synthesis of thiazoles (7a,b)
To a suspension of appropriate thiazole 6a,b (0.38 mL) in DCM (5 mL) trifluoroacetic acid (7.0
mmol, 0.54 mL) was added and the mixture was heated under reflux for 24 h. After cooling, the
mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution and extracted
with dichloromethane (3 x 20). The organic phases was dried (Na2SO4), evaporated under reduced
pressure, and the residue was recrystallized with ethanol to afford the desidered thiazoles 7a,b.
5.1.6.1 3-[2-(1H-Indol-3-yl)-1,3-thiazol-4-yl]-1-(2-methoxyethyl)-1H-pyrrolo[2,3-b]pyridine (7a)
Green solid; yield: 72%; mp: 152 °C; IR cm-1: 3408 (NH); H NMR (200 MHz, DMSO-d6) δ: 3.28
1
(s, 3H, CH3), 3.79 (t, 2H, J = 5.4 Hz, CH2), 4.55 (t, 2H, J = 5.4 Hz, CH2), 7.22-7.32 (m, 3H, H-5’,
H-5’’ and H-6’), 7.48-7.56 (m, 1H, H-7’), 7.73 (s, 1H, H-5), 8.18 (d, 1H, J = 2.8 Hz, H-2’), 8.21 (s,
1H, H-2’’), 8.32-8.38 (m, 2H, H-4’ and H-6’’), 8.66 (dd, 1H, J = 7.9, 1.5 Hz, H-4’’), 11.80 (bs, 1H,
13
NH); C (50 MHz, DMSO-d6) δ: 43.9 (t), 58.0 (q), 70.5 (t), 107.2 (d), 109.2 (s), 110.4 (s), 112.2
(d), 116.3 (d), 118.0 (s), 120.3 (d), 120.8 (d), 122.4 (d), 124.2 (s), 126.8 (d), 128.6 (d), 129.8 (d),
136.6 (s), 141.9 (d), 146.4 (s), 148.8 (s), 162.3 (s). Anal. Calcd for C21H18N4OS: C, 67.36; H, 4.85;
N, 14.96. Found: C, 67.21; H, 4.69; N, 14.74. MS m/z 375 (MH+). HRMS: [MH]+, found 375.1260
C21H18N4OS requires 375.1274.
5.1.6.2
3-[2-(5-Bromo-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-(2-methoxyethyl)-1H-pyrrolo[2,3-
b]pyridine (7b)
1
Green solid; yield: 60%; mp: 128-129 °C; IR cm-1: 3377 (NH); H NMR (200 MHz, DMSO-d6) δ:
3.31 (s, 3H, CH3), 3.79 (t, 2H, J = 5.2 Hz, CH2), 4.55 (t, 2H, J = 5.2 Hz, CH2), 7.28 (dd, 1H, J =
7.9, 4.7 Hz, H-5’’), 7.39 (dd, 1H, J = 8.7, 1.9 Hz, H-6’), 7.50 (d, 1H, J = 8.7 Hz, H-7’), 7.74 (s, 1H,
H-5), 8.18 (s, 1H, H-2’’), 8.24 (s, 1H, J = 2.8 Hz, H-2’), 8.37 (dd, 1H, J = 4.7, 1.4 Hz, H-6’’), 8.54
(d, 1H, J = 1.9 Hz, H-4’); 8.66 (dd, 1H, J = 7.9, 1.4 Hz, H-4’’), 11.98 (bs, 1H, NH); 13C (50 MHz,
DMSO-d6) δ: 43.7 (t), 58.0 (q), 70.6 (t), 107.4 (d), 109.1 (s), 110.1 (s), 113.3 (s), 114.3 (d), 116.3
(d), 117.9 (s), 122.7 (d), 124.9 (d), 126.0 (s), 128.1 (d), 128.3 (d), 129.4 (d), 135.3 (s), 142.2 (d),
146.6 (s), 149.2 (s), 161.7 (s). Anal. Calcd for C21H17BrN4OS: C, 55.64; H, 3.78; N, 12.36. Found:
C, 55.52; H, 3.68; N, 12.60. MS m/z 453 (MH+). HRMS: [MH]+, found 453.0398